Crop breeding aims to generate pure in bred lines with multiple desired traits. Doubled haploid (DH) and genome editing using CRISPR/Cas9 are two powerful game-changing technologies in crop breeding. However, both of ...Crop breeding aims to generate pure in bred lines with multiple desired traits. Doubled haploid (DH) and genome editing using CRISPR/Cas9 are two powerful game-changing technologies in crop breeding. However, both of them still fall short for rapid generation of pure elite lines with integrated favorable traits. Here, we report the development of a Haploid-Inducer Mediated Genome Editing (IMGE) approach, which utilizes a maize haploid inducer line carrying a CRISPR/Cas9 cassette targeting for a desired agronomic trait to pollinate an elite maize in bred line and to generate genome-edited haploids in the elite maize background. Homozygous pure DH lines with the desired trait improvement could be generated within two generations, thus bypassing the lengthy procedure of repeated crossing and backcrossing used in conventional breeding for integrating a desirable trait into elite commercial backgrounds.展开更多
Studies of barley and maize indicate that chromosome doubling occurs via nuclear fusion during an early stage of microspore embryogenesis, but the time and mechanism by which chromosome doubling occurs in bread wheat ...Studies of barley and maize indicate that chromosome doubling occurs via nuclear fusion during an early stage of microspore embryogenesis, but the time and mechanism by which chromosome doubling occurs in bread wheat (Triticum aestivum) remains undetermined. The purpose of this study was to determine the relative time during induction culture when chromosome doubling may occur in wheat, and to identify early indicators for doubled haploid microspores. Microspore nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI) and observed under a fluorescent microscope on the day of isolation, three days after isolation, and six days after isolation. The change in the percentage of microspores containing a single small nucleus, two small nuclei, a single enlarged nucleus, and three or more nuclei was then tracked throughout the six-day period. Ploidy levels were estimated by determining the cross-sectional area and number of nucleoli in microspores containing small and large nuclei then comparing the results of each respective cell-type. The percentage of microspores containing enlarged nuclei increased throughout the six-day test period, and the percentage of binucleated microspores containing small nuclei decreased. Comparison of the changes in average percentage of microspores containing a single small nucleus, binucleated microspores, microspores containing a single large nucleus, and multinucleate microspores on days 0, 3, and 6 indicates that nuclei classified as “small” are likely haploids and nuclei classified as “large” are doubled haploids. The percentage of microspores with enlarged nucleus (nuclei) during the first six days of induction culture could be used as an early indicator for the frequency of chromosome doubling in wheat microspore culture.展开更多
基金National Key R&D Program of China (2016YFD0100303 and 2016YFD0101001)Beijing Natural Science Foundation (6172032).
文摘Crop breeding aims to generate pure in bred lines with multiple desired traits. Doubled haploid (DH) and genome editing using CRISPR/Cas9 are two powerful game-changing technologies in crop breeding. However, both of them still fall short for rapid generation of pure elite lines with integrated favorable traits. Here, we report the development of a Haploid-Inducer Mediated Genome Editing (IMGE) approach, which utilizes a maize haploid inducer line carrying a CRISPR/Cas9 cassette targeting for a desired agronomic trait to pollinate an elite maize in bred line and to generate genome-edited haploids in the elite maize background. Homozygous pure DH lines with the desired trait improvement could be generated within two generations, thus bypassing the lengthy procedure of repeated crossing and backcrossing used in conventional breeding for integrating a desirable trait into elite commercial backgrounds.
文摘Studies of barley and maize indicate that chromosome doubling occurs via nuclear fusion during an early stage of microspore embryogenesis, but the time and mechanism by which chromosome doubling occurs in bread wheat (Triticum aestivum) remains undetermined. The purpose of this study was to determine the relative time during induction culture when chromosome doubling may occur in wheat, and to identify early indicators for doubled haploid microspores. Microspore nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI) and observed under a fluorescent microscope on the day of isolation, three days after isolation, and six days after isolation. The change in the percentage of microspores containing a single small nucleus, two small nuclei, a single enlarged nucleus, and three or more nuclei was then tracked throughout the six-day period. Ploidy levels were estimated by determining the cross-sectional area and number of nucleoli in microspores containing small and large nuclei then comparing the results of each respective cell-type. The percentage of microspores containing enlarged nuclei increased throughout the six-day test period, and the percentage of binucleated microspores containing small nuclei decreased. Comparison of the changes in average percentage of microspores containing a single small nucleus, binucleated microspores, microspores containing a single large nucleus, and multinucleate microspores on days 0, 3, and 6 indicates that nuclei classified as “small” are likely haploids and nuclei classified as “large” are doubled haploids. The percentage of microspores with enlarged nucleus (nuclei) during the first six days of induction culture could be used as an early indicator for the frequency of chromosome doubling in wheat microspore culture.
